The need for replacement of world matrix of fuels based on fossil sources by renewable alternatives made the production of second-generation fuels, for example, ethanol, one of the most promising technologies in development phase. This process consists of conversion of polymers which form the vegetal biomass, mainly those present in cellular wall as cellulose, hemicellulose and lignin, into biofuels and/or biochemicals.
The vegetal biomass is a complex mixture of chemically distinct compounds which can be fractionated generating components with specific applications. Thus, in the same way a petrochemical refinery produces a great variety of products derived from crude oil, the same principles can be applied to biorefineries, that is, refineries based on biomass (Santos, L. V.; Pereira, G. A. G. Petroquimica verde—Anais do Simpésio Microrganismos em Agroenergia: da Prospecçāo aos Bioprocessos. Editora Embrapa. ISSN 2177-4439, 2013).
Although the use of vegetal biomass as a source of fermentable sugars is a promising and sustainable alternative, some challenges need to be overcome, like the availability of sugar from vegetal cellular wall. This procedure may be done through the action of hydrolytic enzymes (cellulases and hemicellulases), which provide the monomers of sugars (hexoses and pentoses) which are posteriorly metabolized by microorganisms for generating biochemicals and biofuels.
However, microorganisms naturally able to consume sugars present in the cellulose and hemicellulose chains, generally, are not efficiently usable in industrial scale. Thus, it is necessary to develop microorganisms with ability to use efficiently these sugars of vegetal cellular wall in industrial scale, as described in the present invention.
The use of microorganisms as efficient platforms in the conversion of biomass sugar into high added value products is widely described. In this sense, the yeast Saccharomyces cerevisiae has received prominent role due to its robustness and tolerance in industrial fermentation conditions. The ease of genetic manipulation of this organism and the use of metabolic engineering tools, in synergy with biology of systems and synthetic biology, has allowed the inclusion of new metabolic routes for producing fuels and chemicals such as ethanol, biobutanol, biodiesel, 1,2-propanediol, succinic acid, pyruvic acid, among others [Cellular and Molecular Life Sciences, 69(16):2671-90, 2012].
Wild lines of S. cerevisae are not naturally able to ferment pentoses, such as, for example, xylose, present in biomass. However, several studies have already done procedures of metabolic engineering in S. cerevisiae through the introduction in these organisms of metabolic routes for consumption of xylose, focusing on two main routes: the Xylose Reductase route—Xylitol Dehydrogenase (XR-XDH) and Xylose Isomerase (XI) route.
Among the studies carried out, the introduction of gene which encodes the xylose isomerase (XI) enzyme allows the strain to present higher yield in the production of alcohol and/or acids than when it is modified with other gene, as for example, gene encoding xylose reductase or xylitol dehydrogenase, since there is less accumulation of undesirable byproducts, such as xylitol and glycerol [2004, FEMS Yeast Res. 4: 655-664].
The XR-XDH route, present in microorganisms eukaryotes, consists of two redox reactions, where xylose is reduced to xylitol by the action of xylose reductase (XR) enzyme, in a reaction mediated by NADPH/NADH and then, xylitol is oxidized to xylulose through the xylitol dehydrogenase (XDH) enzyme, exclusively mediated by NAD+. The NADPH co-factor is mainly regenerated in the oxidative phase of the pentose phosphate route, producing CO2. In addition, NAD+ is regenerated mainly in the respiratory chain, with the O2 as final acceptor of electrons. Under limited oxygen concentrations, the complete reoxidation of NAD+ does not occur, resulting in a redox unbalance and the accumulation of xylitol, which directly impacts the final yield of ethanol [Biochemical Engineering Journal, Amsterdam, v. 12, n. 1, p. 49-59, 2002]. In addition to xylitol, other byproduct formed is glycerol, due to re-oxidation of excess NADH through XDH [FEMS Yeast Research, Delft, v. 4, n. 6, p. 655-664, 2004].
The xylose isomerase (XI) route, more common in prokaryotes, occurs in a single step, avoiding redox unbalance and the formation of byproducts that decrease the yield of ethanol. For several decades, attempts at heterologous expression of bacterial XI in S. cerevisiae were not successful [Enzyme and Microbial Technology, Amsterdam, v. 32, n. 2, p. 252-259, 2003]. In 2003, the functional expression in S. cerevisiae of a xylose isomerase of anaerobic fungus Piromyces sp. [FEMS Yeast Research, Delft, v. 4, n. 1, p. 69-78, 2003] and in 2009 of Orpiromyces sp. fungus [Applied Microbiology and Biotechnology, Heidelberg, v. 82, n. 6, p. 1067-1078, 2009], resulted in mutants able to grow in xylose as the only source of carbon, with high activities of these enzymes, higher yield in the production of ethanol, lower production and accumulation of intermediate metabolites and with less catabolic repression in medium containing glucose and xylose [FEMS Yeast Research, Delft, v. 4, n. 6, p. 655-664, 2004; FEMS Yeast Research, Delft, v. 5, n. 4, p. 399-409, 2005a; FEMS Yeast Research, Delft, v. 5, n. 10, p. 925-934, 2005b]. The XR-XDH route has higher initial productivity for producing ethanol more rapidly, only with the insertion of the genes responsible for conversion of xylose, however, the XI route has a higher yield for not accumulating byproducts [Microbial Cell Factories, Londres, v. 6, n. 5, p. 1-10, 2007].
Many documents, as, for example, W02006/009434, WO2011/153516, U.S. Pat. No. 8,399,215, EP2679686 and WO2014018552 describe microorganisms able to use pentoses, more specifically xylose, as a source of carbon. In order to be able to consume xylose, it is necessary that the microorganism is genetically modified at least with the addition of the gene encoding xylose isomerase. As a strategy to improve yeast productivity, the genes encoding Xylulokinase and the genes of the pentoses phosphate route can be overexpressed: Transketolase, ribose 5-phosphate isomerase, ribose 5-phosphate epimerase and Transaldolase. Furthermore, the inactivation of the gene encoding an aldose reductase (GRE3) can be performed, aiming for a lower accumulation of xylitol and a higher yield of ethanol.
Therefore, the literature shows that increased expression of the genes described above that favor the conversion of xylose to ethanol is necessary for the consumption of this sugar is efficient. Thus, the microorganisms described in the prior art, which were genetically modified for consumption of xylose, can have (but not necessarily) the genetic modifications described above. What basically differentiates the efficiency and productivity in the anaerobic conversion of xylose in biofuels and/or biochemicals presented by each one of these is the form and location how these genes are incorporated to the microorganism genome, considering the best possible combination between these genes and respective promoters by which they are regulated, as well as the appropriate choice by the sequence of nucleotides encoding xylose isomerase, being this the main gene that, when expressed, enables the consumption of xylose for each modified microorganism, in addition to adaptation of microorganism through evolution. Thus, the present invention shows advantageously better yield and productivity than the microorganisms previously described in the prior art.
The present invention describes, among other objects, a genetically modified microorganism for inclusion of genes of the pentose phosphate route, as well as those of Xylulokinase, and inactivation of the aldose reductase gene, as described in the documents WO2006/009434, WO2011/153516, U.S. Pat. No. 8,399,215, EP2679686 and WO2014018552. The genetically modified microorganism of the present invention differs advantageously from the previous one by the fact that the genes have been more efficiently combined with their promoters, as well as inserted into more convenient location in the microorganism genome, when compared to the previously mentioned documents. Additionally, the gene encoding xylose isomerase herein inserted has been optimized, by the inventors, for the preferably codons of microorganism in which it was first inserted, in this case, the microorganism of Saccharomyces cerevisiae species. In the present document, the genes are inserted into the microorganism through the homologous recombination, then going to interact with its genome.
Specifically, referring to the production of ethanol, the obtaining of some lines able to act in industrial scale was successful. However, such strains are still susceptible to have their fermentative performance compromised or even be replaced by wild lines when subjected to the stressful conditions of the Brazilian process of ethanol production.
In the Brazilian fermentative process for ethanol production, it is usual that the plants do the intensive reuse of yeast cells used in the fermentation, process known as recycling. In this process, up to 90% of yeasts may be reused from one fermentation to another, resulting in very high cell densities inside the fermenter and making the fermentation time very short [FEMS yeast research, 8(7):1155-63, 2008].
In some industrial pants, the recycling can occur throughout the period harvest, lasting up to nine months. Thus, the prolonged period of recycling added to continuous input of microbial contaminants into the system, makes the fermentation environment highly competitive, imposing severe biotic and abiotic tensions on the strains of yeast used in the process [International Sugar Journal, Londres, v. 112, p. 86-89, 2010]. This competitive environment results in the replacement of yeasts which started the fermentation process by wild yeasts. This fact happens because the wild yeasts naturally occur in sugarcane and, therefore, are inserted together with it in the process of fermentation, thus ending up contaminating the entire industrial process [FEMS yeast research, 8(7):1155-63, 2008].
Additionally, some studies demonstrated that those yeasts that started the fermentation process, and ended up being replaced by the wild, are also not able to survive stressful situations of the industrial process of fermentation, such as high concentration of ethanol, high temperature, osmotic stress due to salts and sugars, acidity, sulfites and bacterial contamination [FEMS yeast research, 8(7):1155-63, 2008]. Thus, the obtaining of line with efficient capacity of resistance to the aggressive industrial process of fermentation, as well as susceptible to genetic modifications to acquire characteristics of interest, such as the consumption of pentoses, more specifically xylose, is not a trivial process.
The microorganism described in the present invention, therefore, is advantageously adapted to the Brazilian process of industrial fermentation and is shown to be differentially efficient in the conversion of sugars from vegetal biomass, mainly lignocellulosic material, to biofuels and/or biochemicals, that is, with sufficient yield to be applied in industrial scale, even under the stressful conditions of the Brazilian fermentation process.
Additionally, the microorganism described in the present invention shows features of industrial interest such as: being a non-flocculating strain, presenting high yield of ethanol, low formation of glycerol and xylitol, high viability, high growth rate, non-production of foam, among others.